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Biochemical Verification of Tobacco Use and Abstinence: 2019 Update
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BACKGROUND: The changing prevalence and patterns of tobacco use, the advent of novel nicotine delivery devices, and the development of new biomarkers prompted an update of the 2002 Society for Research on Nicotine and Tobacco (SRNT) report on whether and how to apply biomarker verification for tobacco use and abstinence. METHODS: The SRNT Treatment Research Network convened a group of investigators with expertise in tobacco biomarkers to update the recommendations of the 2002 SNRT Biochemical Verification Report. RESULTS: Biochemical verification of tobacco use and abstinence increases scientific rigor and is recommended in clinical trials of smoking cessation, when feasible. Sources, appropriate biospecimens, cutpoints, time of detection windows and analytic methods for carbon monoxide, cotinine (including over the counter tests), total nicotine equivalents, minor tobacco alkaloids, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol are reviewed, as well as biochemical approaches to distinguishing cigarette smoking from use of electronic nicotine delivery devices (ENDS). CONCLUSIONS: Recommendations are provided for whether and how to use biochemical verification of tobacco use and abstinence. Guidelines are provided on which biomarkers to use, which biospecimens to use, optimal cutpoints, time windows to detection, and methodology for biochemical verifications. Use of combinations of biomarkers is recommended for assessment of ENDS use. IMPLICATIONS: Biochemical verification increases scientific rigor, but there are drawbacks that need to be assessed to determine whether the benefits of biochemical verification outweigh the costs, including the cost of the assays, the feasibility of sample collection, the ability to draw clear conclusions based on the duration of abstinence, and the variability of the assay within the study population. This paper provides updated recommendations from the 2002 SRNT report on whether and how to use biochemical markers in determining tobacco use and abstinence.
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“authors like Benowitz et al. (2020) and West et al. (2005) suggest that abstinence outcomes are also reliable in those studies in which biochemical verification is not feasible, such as those in which the data are collected by phone or through the internet or when personal contact is limited.”
From Evaluation of effectiveness and acceptability of a psychological treatment for smoking cessation combined with a smartphone App: A pilot study (2024) · cites this paper as Benowitz et al., 2020
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“—Self-reported smoking status four weeks after the scheduled quit attempt was verified using expired carbon monoxide (CO) levels (Benowitz et al., 2019).”
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“12 In many clinical, epidemiological and genome wide association studies serum cotinine levels are used to access the relationship of tobacco use to the outcome of interest, be that tobacco dependence or disease risk (1).”
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“Its blood half-life is approximately 2 hours, and therefore, it is not a useful marker for assessing smoking abstinence [13].”
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“Following recommendations, self-reported abstinence was verified using objective biochemical methods (in this study, initially expired CO and subsequently cotinine in urine) [21].”
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“Based on a receiver operation characteristics (ROC) analysis, optimal thresholds for NNAL were calculated to minimize the difference between sensitivity and specificity considering NNAL compliance boundaries at 10, 25 and 40 pg/mL.(12) All analyses were performed with SAS version 9.”
BackgroundMethodInfluentialJiang, N., Zhao, A., Rogers, E. S., Cupertino, A. P., Zhao, X., Cartujano-Barrera, F., Siu, K., & Sherman, S. E. (2024) · JMIR mHealth and uHealth · doi:10.2196/59496 · Cited by 5
“A CO concentration of ≤ 6 parts per million indicated biochemically verified abstinence [26].”
BackgroundPredescu, I.-A., Jîjie, A.-R., Pătrașcu, D., Pasc, A.-L.-V., Piroș, E. L., Trandafirescu, C. M., Oancea, C. I., Dehelean, C. A., & Moacă, E.-A. (2025) · Pharmacy · doi:10.3390/pharmacy13010007 · Cited by 7
Mahabee‐Gittens, E. M., Matt, G. E., López-Gálvez, N. I., Hoh, E., Quintana, P. J. E., Dodder, N. G., Jandarov, R., Stone, L., Wullenweber, C. A., Ahluwalia, J. S., et al. (2025) · Pediatric Research · doi:10.1038/s41390-025-03915-3 · Cited by 5
“We recruited 0 – 11-year-old children and their parents and used strict parental screening and biochemical validation techniques using cotinine, the main nicotine metabolite, 26 and hand wipe nicotine, a THS pollutant, 2,3,18,22 to categorize and compare children in three TSE groups ( N = 1013).”
Küçükaksu, M. H., Jansen, L., Hoekstra, T., Helmig, S., Adriaanse, M. C., & van Meijel, B. (2025) · PLoS ONE · doi:10.1371/journal.pone.0322160 · Cited by 5
“Generally, CO monitoring is used in research to biochemically verify smoking status with the outcome in parts per million (ppm) [34,35].”
Heffner, J. L., Baker, K., Georgiou, K., Graham, A. L., Kelly, M. M., Konstantinou, P., Lamprou, E., Lele, C., Lok, K. Z., Orzechowski, M., et al. (2025) · Nicotine & Tobacco Research · doi:10.1093/ntr/ntaf112 · Cited by 5
“…testing, since cotinine cannot distinguish e-cigarette and other tobacco use from NRT use and the only method that can potentially make this dis-tinction—urinary analysis of minor tobacco alkaloids (eg, anatabine/anabasine) 27 —was not feasible in a remote trial de-sign with national recruitment.”
Shelley, D., Armstrong-Hough, M. J., Nguyen, T., Alvarez, G. G., Kapur, R., Shuter, J., Goldsamt, L. A., Tozan, Y., Minh, H. V., Vu, G. V., et al. (2026) · The Lancet Global Health · doi:10.1016/s2214-109x(25)00451-6 · Cited by 5
Lee, J. H., Shim, J., & Lee, J. (2023) · Korean Journal of Family Medicine · doi:10.4082/kjfm.22.0153 · Cited by 5
Colsoul, M., Goderniaux, N., Onorati, S., Dupuis, S., Jamart, J., Vanpee, D., Berlin, I., & Galanti, L. M. (2023) · Nicotine & Tobacco Research · doi:10.1093/ntr/ntad130 · Cited by 5
Xie, X., Liang, L., Nan, Y., Zhang, L., & Xiao, L. (2024) · BMC Public Health · doi:10.1186/s12889-023-16496-9 · Cited by 5
“Considering there is no close contact or interaction between participants and the study team, biochemical verification may be infeasible as the SRNT guidelines have suggested [35], it will be self-reported.”
BackgroundKunutsor, S. K., Tetteh, J., Dey, R. S., Touw, D. J., Dullaart, R. P. F., & Bakker, S. J. L. (2024) · Primary care diabetes · doi:10.1016/j.pcd.2024.04.004 · Cited by 5
“For a more precise measurement, attention has been focused on objective markers of smoking exposure including cotinine [9].”
Biosynthesized gold nanoparticles for enhanced determination of nicotine in heated tobacco products
Xue, Y., Wu, Y. L., Zhang, X., Yan, J., Long, Z., Xi, F., Luo, D., Liu, Q., Guo, L., Jing, Y., et al. (2024) · International Journal of Electrochemical Science · doi:10.1016/j.ijoes.2024.100721 · Cited by 5
Outcomes of a Comprehensive Mobile Vaping Cessation Program in Adults Who Vape Daily: Cohort Study
Marler, J. D., Fujii, C. A., Utley, M. T., Balbierz, D. J., Galanko, J. A., & Utley, D. S. (2024) · JMIR Formative Research · doi:10.2196/57376 · Cited by 7
“Biovalidation in remote digital vaping cessation studies presents various challenges, including distinguishing between use of e-cigarettes and other sources of nicotine, cost, logistics, resource use, and participant compliance in sample collection [47,48].”
BackgroundMckeon, K. L., Werthmann, D., Straubing, R., Rodriguez, A., Sosnoff, C. S., Blount, B. C., Chew, G. L., Reponen, T. A., Adamkiewicz, G., Hsu, J., et al. (2024) · Journal of Clinical and Translational Science · doi:10.1017/cts.2024.581 · Cited by 5
Quitline-Based Young Adult Vaping Cessation: A Randomized Clinical Trial Examining NRT and mHealth
Vickerman, K. A., Carpenter, K., Hunter‐Mullis, K., Shoben, A. B., Nemeth, J. M., Mayers, E., & Klein, E. G. (2024) · American Journal of Preventive Medicine · doi:10.1016/j.amepre.2024.10.019 · Cited by 5
Cui, G., Jiao, X., Wang, Z., & Zhang, Z. (2025) · Frontiers in Public Health · doi:10.3389/fpubh.2025.1502341 · Cited by 5
von Weymarn, L. B., Thomson, N. M., Marchand, L. L., & Murphy, S. E. (2025) · Chemical Research in Toxicology · doi:10.1021/acs.chemrestox.5c00310 · Cited by 5
Berveiller, P., Rault, E., & Guerby, P. (2020) · Gynécologie Obstétrique Fertilité & Sénologie · doi:10.1016/j.gofs.2020.03.023 · Cited by 5
Valencia, S., Callinan, L. S., Shic, F., & Smith, M. V. (2020) · JMIR mhealth and uhealth · doi:10.2196/18809 · Cited by 5
“Prior work has suggested a variety of optimal cutoff breath CO levels to determine smoking status [12,24,30-32].”
BackgroundGuydish, J. R., Straus, E. R., Le, T. L., Gubner, N. R., & Delucchi, K. (2020) · Tobacco Control · doi:10.1136/tobaccocontrol-2020-056000 · Cited by 5
Allen, A. M., Skobic, I., Bell, M. L., Medvescek, K., ALLEN, S. S., Collins, B. N., & Nair, U. S. (2021) · Addictive Behaviors · doi:10.1016/j.addbeh.2021.107153 · Cited by 5
Objective measure of smoking status highlights disparities by sex
Khadanga, S., Yant, B., Savage, P. D., Rengo, J. L., & Gaalema, D. E. (2022) · American Heart Journal Plus Cardiology Research and Practice · doi:10.1016/j.ahjo.2022.100171 · Cited by 5
“One way to objectively measure recent smoking is through the amount of carbon monoxide (CO) (a gas that is a byproduct of incomplete combustion) in expired breath [6].”
BackgroundHe, L., & Xi, X. (2022) · BMC Pediatrics · doi:10.1186/s12887-022-03571-0 · Cited by 5
“Serum cotinine levels can reflect the passive exposure of children to cigarettes, which is the main metabolite of nicotine with a long half-life, about 15–20 h [8, 9].”
BackgroundPoudel, K. C., Poudel‐Tandukar, K., Silwal, R. C., Chalise, B. S., Bertone‐Johnson, E. R., & Vidrine, D. J. (2023) · AIDS and Behavior · doi:10.1007/s10461-023-04062-8 · Cited by 5
“Participants who self-report abstinence but had a CO level of ≥ 5ppm were considered smokers [32].”
BackgroundAdkins-Hempel, M., Japuntich, S. J., Chrastek, M., DUNSIGER, S. I., Breault, C., Ayenew, W., Everson‐Rose, S. A., Nijjar, P. S., Bock, B. C., Wu, W., et al. (2023) · Addiction Science & Clinical Practice · doi:10.1186/s13722-023-00388-9 · Cited by 6
“For those using nicotine replacement or noncombustible tobacco products, a carbon monoxide (CO) breath sample will be used to verify abstinence (CO < 6 ppm will indicate abstinence [61, 63]).”
MethodSifat, M. S., Hébert, E. T., Ahluwalia, J. S., Businelle, M. S., Waring, J. J. C., Frank-Pearce, S. G., Bryer, C. M., Benson, L., Madison, S. D., Planas, L. G., et al. (2023) · JMIR Formative Research · doi:10.2196/48857 · Cited by 6
“…Vitalograph ecolyzer (Vitalograph Inc; for in-clinic assessments) or via the Bedfont iCO Smokerlyzer (Bedfont Scientific Ltd; for remote smartphone-based assessments) with a CO level of ≤ 6 ppm indicating biochemical confirmation of self-reported abstinence (per current recommendations [35]).”
BackgroundMethodFahey, M. C., Krukowski, R. A., Anderson, R. T., Cohn, W. F., Porter, K., Reid, T., Wiseman, K. P., You, W., Wood, C. H., Rücker, T., et al. (2023) · Contemporary Clinical Trials · doi:10.1016/j.cct.2023.107335 · Cited by 5
Gao, T., Hou, M., Wang, Q., Liu, D., Chen, F., Xing, Y., & Mei, J. (2023) · Frontiers in Nutrition · doi:10.3389/fnut.2023.1285494 · Cited by 5
Luo, Y., Zhang, L., Lu, Y., Lin, X., Weng, Z., & Xu, Y. (2023) · Biological Trace Element Research · doi:10.1007/s12011-023-04037-9 · Cited by 5
“Coti-nine is a highly specific metabolite of nicotine, the primary addictive chemical in tobacco, and is used as a biomarker of environmental tobacco smoke exposure [16].”
Krotter, A., Pérez, Á. G., Diego, G. A., & García–Fernández, G. (2024) · Eating Behaviors · doi:10.1016/j.eatbeh.2024.101882 · Cited by 5
Siddiqi, K., Welch, C., Huque, R., Iqbal, R., Kanaan, M. N., Mishu, M. P., Khokhar, M. A., Semple, S. E., CLASS III Trial Team, Sheikh, A., et al. (2024) · Nicotine & Tobacco Research · doi:10.1093/ntr/ntae130 · Cited by 6
Orfin, R. H., Ramos-Santiago, J. W., Soriano, R. D., Acosta, E. R., Bermudez, D., Rodriguez, Y. L., Li, D., Rahman, I., McIntosh, S. W., Ossip, D. J., et al. (2024) · Journal of Ethnicity in Substance Abuse · doi:10.1080/15332640.2024.2397422 · Cited by 5
“However, self-reported abstinence has been judged to be appropriate for minimal contact interventions (Benowitz et al., 2020).”
Watson, N. L., Mull, K. E., & Bricker, J. B. (2020) · Drug and Alcohol Dependence · doi:10.1016/j.drugalcdep.2020.108394 · Cited by 5
“However, verifying abstinence is often considered unnecessary in population-level intervention studies with no face-to-face contact as doing so is often not feasible and can bias results (Benowitz et al., 2020; Benowitz et al., 2002).”
BackgroundRamos, G. G., Sussman, S. Y., Moerner, L., Unger, J. B., & Soto, C. (2022) · Journal of Drug Education · doi:10.1177/00472379221111542 · Cited by 5
Li, Y., & Hecht, S. S. (2022) · Chemical Research in Toxicology · doi:10.1021/acs.chemrestox.2c00195 · Cited by 5
Self-citationReyes, G. D. L., Ng, A., Chavez, J. V., Apollonio, D. E., Kroon, L. A., Lee, P. L., & Vijayaraghavan, M. (2023) · Substance Use & Misuse · doi:10.1080/10826084.2023.2231060 · Cited by 5
Wróblewski, K., Szultka‐Młyńska, M., Courtney, R. J., Buszewski, B., & Tutka, P. (2023) · International Journal of Molecular Sciences · doi:10.3390/ijms242015364 · Cited by 5
“The results of our study con fi rm the usefulness of LC-Q-TOF-MS in quanti fi cation and represent a new perspective of the utility of LC-HRMS systems in the future.”
Background
Metadata
- Authors
- Benowitz, N. L., Bernert, J. T., Foulds, J., Hecht, S. S., Jacob, P., Jarvis, M. J., Joseph, A., Oncken, C., & Piper, M. E.
- Affiliations
- University of California, San Francisco (US)
- Centers for Disease Control and Prevention (US)
- Pennsylvania State University (US)
- University of Minnesota (US)
- University College London (GB)
- University of Connecticut (US)
- University of Wisconsin–Madison (US)
- Corresponding author
- Benowitz, Neal L
- ORCID
- Venue
- Nicotine & Tobacco Research
- Publisher
- Oxford University Press (OUP)
- Published
- 2019-10-01
- Volume
- 22
- Issue
- 7
- Pages
- 1086-1097
- ISSN
- 1462-2203 (ISSN-L)
- 1469-994X
- PMC
- PMC7882145
- OpenAlex
- W2978389675
- Cited by
- 619
- References
- 121
- Citations by year
- 2026: 99 · 2025: 84 · 2024: 107 · 2023: 103 · 2022: 87 · 2021: 79 · 2020: 54 · 2019: 4
- FWCI
- 6.58
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model (vor)
- Locations
- Nicotine & Tobacco Research (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- National Institutes of Health (CC999999)
- Topics
- Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Smoking Behavior and Cessation
- Health Sciences › Medicine › Epidemiology › Substance Abuse Treatment and Outcomes
- Health Sciences › Medicine › Pediatrics, Perinatology and Child Health › Prenatal Substance Exposure Effects
- Keywords
- tobacco use, smoking abstinence, biomarkers, cotinine, carbon monoxide, electronic cigarettes, NNAL, biological specimen, detection window
- MeSH terms
- Cigarette Smoking, Carbon Monoxide, Cotinine, Humans, Nicotine, United States, Biomarkers, Smoking Cessation, Tobacco Products
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